Ординатура / Офтальмология / Английские материалы / Essentials in Ophthalmology Cornea and External Eye Disease_Reinhard_Larkin_2005

Fig. 13.50 A–D. Schematic illustration (A) and 3-D reconstruction (B–D) of the corneal epithelium with anterior stroma and nerves (healthy human subject): B anterior view, C posterior view, D anterior view with

currently thought to be the maximum when patient and examiner movements are taken into account.

Images are presented in the form of a series of 2-D grayscale images (384¥384 pixels, 8 bit) representing optical sections through the cornea. The original raw image stacks were converted using ImageJ (NIH, USA) for 3-D reconstruction using Amira 3.1 (TGS Inc., USA). The voxel size is around 0.8¥0.8¥0.9 mm using the above-mentioned acquisition parameters. The Amira volume-rendering software package provides an interactive environment allowing features such as volume orientation for viewing planes and 3-D perspectives, segmentation and determination of distances and surfaces. The image stacks were carefully aligned and modified to eliminate unspecific information by adapting the gray values in the depicted spec-

virtual removal of the epithelium. Thin nerves running parallel to Bowman’s membrane in the basal epithelial plexus. Thicker fibers originating from the subepithelial plexus

trum. Shadows and illumination were manipulated after assigning density values to gray values to more clearly visualize the spatial arrangement without loss of information.

As a first in vivo application of the new device in combination with 3-D reconstruction techniques, nerve fiber distribution was characterized in healthy human corneal epithelium. The spatial arrangement of epithelium, nerves and keratocytes was visualized by in vivo 3-D confocal laser-scanning microscopy (CLSM) (Fig. 13.50). The 3-D reconstruction of the cornea in healthy volunteers yielded a picture of the nerves in the central part of the human cornea. Thick fibers arise from the subepithelial plexus, and the nerves further subdivide diand trichotomously, resulting in five to six thinner fibers arranged parallel to Bowman’s membrane and with partial interconnections

Fig. 13.51. Nerve fibers of the basal epithelial plexus, in strict alignment parallel to Bowman’s membrane

(Fig. 13.51). Branches penetrating the anterior epithelial cell layer cannot be visualized.

In conclusion, 3-D CLSM is the first technique to permit visualization and analysis of the spatial arrangement of the epithelium, nerves and keratocytes in the living human cornea. The method developed provides a basis for further device refinements and for studies of changes in cellular arrangement and epithelial innervation in corneal disease. For example, CLSM may help to clarify gross variations of nerve fiber patterns under various clinical and experimental conditions.

13.6.2

Functional Imaging

In conventional microscopy the possibility of using dyes to visualize specific anatomic structures yields major information gains. This is especially true when techniques of fluorescence microscopy or immunohistochemistry are used in combination with confocal techniques [67]. Because these methods are well suited for investigating the functional status of tissues, they are also interesting for in vivo microscopy in humans, for example, for studies of wound healing or inflammation processes. However, problems arise due to the necessity for “real time” investigation because of involuntary movements on the part of the subjects and due to the selection of suitable non-toxic vital stains. Nevertheless, successful initial steps have already been taken toward confocal in vivo fluorescence microscopy of the anterior eye segments [20, 29], and

A

B

Fig. 13.52 A, B. Fluorescence micrographs of the superficial corneal epithelial cells: A intact corneal epithelium without appreciable fluorescence; B stippled epithelium after contact glass examination, blue marked area with fluorescein-stained cells, orange marked area with unstained intact cells

these may be regarded as a further enhancement of corneal assessments by slit-lamp microscopy following fluorescein or rose bengal staining [68, 17, 16, 42, 52].

To achieve this, a Heidelberg Retina Angiograph (HRA/C, Heidelberg Engineering GmbH, Germany) has been modified with a lens attach-

ment (Rostock Cornea Module) so that the laser focus is shifted to the anterior eye segments. This enables fluorescence microscopy images to be obtained after staining with the non-specific stain sodium fluorescein and excitation with blue argon laser light (wavelength 488 nm) and addition of a barrier filter (500 nm). Using a green argon laser (514 nm) in reflection mode with the same device, it is also possible to visualize break-up phenomena of the tear film [29, 75, 76] (Figs. 13.52–13.54).

The result is a technique that enables fur- ther-reaching investigations of damaged corneal epithelium and of the associated wound healing processes. In future, confocal fluorescence microscopes specially designed for in vivo investigations in humans will perhaps permit highquality functional imaging that is even more comprehensive and specific.

Fig. 13.54 A–D. Same patient as in Fig. 13.52 after application of a local anesthetic and following applanation tonometry: A slit-lamp microscopy photograph with corneal stippling; B reflection mode, tear film

Summary for the Clinician

∑Confocal high-resolution biomicroscopy will be used for the in vivo description of corneal pathology at the cellular level

∑It will enable degeneration and repair mechanisms under various conditions to be examined so that the findings can be correlated with those from conventional slitlamp biomicroscopy

defect (dry spot) just 3 s after eyelid opening; C fluorescence mode, same area as in B, superficial corneal epithelium, area with marked fluorescence; D fluorescence mode, higher magnification

∑This will generate enhanced quality in clinical evaluation

∑The use of vital staining substances, e.g., sodium fluorescein or etidium homodimer or calcein, may give insights into the metabolic activities of a variety of cells under different wound healing or degenerative conditions

Acknowledgements. The authors are grateful for the cooperation and detailed contributions of Alexander Eckard, Steffi Knappe, Robert Kraak,Petra Schröder,Oliver Stachs,Hans-Peter Vick, and Andrej Zhivov.

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|Core Messages

∑Allergic eye disease affects a reported 20 % of the population worldwide and

may be increasing in line with other atopic diseases, such as asthma, as a result of environmental factors

∑Other pathological mechanisms, in addition to the standard type I hypersensitivity reaction, have been recently implicated in the pathogenesis of allergic eye disease

∑Established treatments have targeted mast cells, but as a result of our greater understanding of the mechanisms involved in eye allergy, researchers are now concentrating on other cell types, such as eosinophils and dendritic cells, as potential targets for immunomodulation

∑Other areas of investigation to elucidate novel treatment strategies include the study of the genetics of ocular allergy, the role of environmental factors in the pathogenesis of ocular allergy, and the

use of immunostimulatory DNA sequences that can inhibit the allergic response

14.1 Introduction

Owing to the fact that the eye is one of the first organs to encounter environmental allergens, allergic eye disease has become a common ocular problem, estimated to affect about 20% of the population worldwide [51]. Allergic eye disease is one of a spectrum of diseases that share a common initiating mechanism and pattern of

inflammation and is a problem that is widespread among individuals who suffer with allergies. Although the incidence of allergic eye disease varies by geographical location, its prevalence is difficult to gauge as allergies tend to be underreported.A recent survey conducted by the American College of Allergy,Asthma and Immunology found that 35% of families interviewed in the United States experienced allergies, 50% of whom reported associated eye symptoms [48]. However, this prevalence is set to increase probably as a result of environmental factors. For example,the morbidity and mortality of asthma have increased with this, coinciding with the increase in house dust mite levels, and are greatest in communities exposed to high allergen levels [32].

Geographical variations, the lack of any clear-cut objective diagnostic criteria and the difficulty over the diagnosis – especially when it is the sole manifestation of atopy – have made it difficult to report the incidence rates for different forms of allergic eye disease. In the past, clinical features were used to classify allergic eye disease, but recent work that has defined the underlying pathogenic mechanisms has provided an understanding of the cellular and mediator mechanisms involved, thereby enabling a better understanding of the disease process and the development of more effective treatments.

Allergic conjunctivitis is typically divided into five types: seasonal allergic conjunctivitis (SAC), perennial allergic conjunctivitis (PAC), vernal keratoconjunctivitis (VKC), atopic keratoconjunctivitis (AKC) and giant papillary conjunctivitis (GPC). The latter is an iatrogenic disease associated with foreign bodies on the eye, such as contact lenses and ocular prostheses.

Although not always included in this grouping, it is thought to have a possible allergic mechanism because of the predominance of mast cells. GPC invariably resolves when the cause is removed and keratopathy is rare.

The aim of this review will be to focus on the underlying mechanisms of allergic eye disease and the current classification of the various disease manifestations. Treatment modalities, both well established and new innovations, will also be discussed.

14.2 Pathophysiology

Ocular allergic disease is typically associated with immunoglobulin E mediated mast cell activation (type I immediate hypersensitivity reaction) in the conjunctival tissue. However, recent data from several groups indicate that other additional mechanisms can also be involved in causing a red, allergic eye.

14.2.1

Type I Hypersensitivity

The allergic response begins when allergen is encountered by an antigen presenting cell (APC), either directly or as part of an immune complex with immunoglobulin. The APCs then process and present the allergen to CD4+ T cells as a peptide fragment in association with the major histocompatibility (MHC) class II molecule. These T cells are then polarized into T helper type 1 (Th1) cells and T helper type 2 (Th2) cells. The Th2 cells produce a variety of interleukins, two of which – IL-4 and IL-13 – stimulate immunoglobulin class switching of B cells from producing IgM to producing IgE. This immunoglobulin binds to high affinity receptors (FceRI) on the surface of mast cells and basophils. Subsequent encounter with this allergen results in the cross linkage of IgE bound to FceRI on the surface of mast cells and a cascade of signal transduction with a resultant release of preformed and newly synthesized mediators. Tissue fibroblasts and epithelial cells are also triggered by Th2 cells to produce chemokines

such as monocyte chemoattractant protein-1 (MCP-1), eotaxin-1, or the protein regulated on activation normal T-cell expressed and secreted (RANTES),resulting in the migration of inflammatory cells into the site of allergen exposure [5].

This sensitized mast cell mediated response is responsible for many of the symptoms seen in SAC and PAC – such as itching, redness and eyelid swelling – with most of these patients having a positive family history of atopy and raised levels of allergen specific IgE in the serum and tears [32]. Immunohistochemical studies have shown that in SAC there is a significant increase in the numbers of conjunctival mast cells,which correlates with the patient’s severity of symptoms [32]. A number of proinflammatory cytokines are released by mast cells and these include histamine, leukotriene C4, prostaglandin D2, platelet-activating factor (PAF), tryptase, chymase, cathepsin G and other eosinophil and neutrophil chemoattractants in what is termed the early phase response [32]. This response lasts for a maximum of 20 min after allergen activation and includes enhanced tear levels of histamine, protease tryptase, and leukotrienes, and an increase in the number of eosinophils [46]. At about 6 h a late phase response occurs which includes a second peak of tear histamine (without an increase in tryptase) and an increase in tissue adhesion molecules E-selectin and interstitial cell adhesion molecule 1 (ICAM- 1), which is followed by an influx of inflammatory cells such as neutrophils, T cells, basophils and eosinophils [46]. The presence of tear histamine and the absence of tear tryptase in the late phase response may indicate that basophils, as opposed to mast cells, are involved.

Mast cells are also known to synthesize, store and release a number of cytokines such as IL-4, IL-5, IL-8, IL-13 and TNFa [46]. Cytokine involvement, particularly the Th2 cytokines, has been the focus of many studies recently looking into the mechanisms of ocular allergy. It is known, for example, that IL-4 plays a key role in allergic inflammation by promoting T-cell growth, by inducing the production of IgE from B cells, by upregulating the adhesion molecule vascular cell adhesion molecule 1 (VCAM 1), and by regulating the differentiation of the Th2

subset, which is essential for the allergic reaction [19, 31].

Physiologically,mast cells represent a heterogeneous population. They are subdivided on the basis of their ultrastructural characteristics, protease content, and T-lymphocyte dependency [49]. In humans, mast cells that contain tryptases, chymases, carboxypeptidase A, and cathepsin G are designated MCTC and those that contain tryptase only are designated MCT. Although both subtypes develop from the same CD34+ mononuclear precursor, the MCT subtype is dependent on the presence of T lymphocytes, present at mucosal surfaces, and increases in number in aeroallergen driven allergic disease, whilst the MCTC subtype appears to be independent of T cells but its development requires fibroblastic derived growth factors, which are predominant in connective and perivascular tissues, and is characteristic of fibrotic processes [32]. Normally, approximately 80% of conjunctival mast cells are of the MCTC phenotype and are mainly subepithelial in distribution, with the rest being MCT, but during allergic inflammation such as that seen in SAC, VKC or AKC, the numbers of the latter type increase in the epithelial and subepithelial layers [37]. In the chronic and fibrosing condition AKC, however, the MCTC subtype predominates, perhaps indicating an important transition from a simple mediator driven disorder to that of chronic inflammation leading to conjunctival fibrosis [37].

14.2.2

Ocular Inflammatory Reaction: Late Phase

A late phase reaction sustained by a complex network of inflammatory cells and mediators can also occur in the eye. This has been demonstrated in humans using allergen for conjunctival provocation of allergic subjects [10]. Allergen challenge caused the typical early-phase reaction within 20 min, with the initial reaction being dose dependent. With smaller doses of allergen the reaction was not so pronounced and spontaneous recovery occurred within a brief period.With larger doses, the reaction was

more persistent and progressed to a late-phase reaction. Typically, high doses of allergen induced a continuous reaction manifested by burning, redness, itching, tearing and a foreign body sensation that began 4–8 h after challenge and persisted for up to 24 h. This clinical reaction was accompanied by a significant recruitment of inflammatory cells in tears. Neutrophils first appeared about 20 min after challenge, with eosinophils and lymphocytes increasing in prominence 6–24 h after challenge.

The eosinophil predominates in the late phase reaction. It is a powerful effector cell, releasing arginine rich toxic proteins capable of causing corneal epithelial damage [32]. Normally, eosinophils are not found in the conjunctival epithelium of non-atopic subjects but the numbers are increased in the conjunctival epithelium, subepithelium and tears of patients with AKC and, to a greater extent, VKC patients. Furthermore, this increase in eosinophils and eosinophil products [e.g. eosinophil peroxidase, eosinophil cationic protein (ECP)] is also present in both skin test positive and skin test negative VKC and is not confined to ocular tissues.This suggests that,in at least some forms of allergic conjunctivitis such as VKC,eosinophilic infiltration – and not IgE sensitization – is the more relevant feature of the disease and is associated with signs of systemic activation of eosinophils [10].

14.2.3

Non-specific Conjunctival Hyperreactivity

Non-specific stimuli can also cause target organ hyperreactivity and this is thought to play a role in allergic diseases of the eye. It is postulated that“non-specific conjunctival hyperreactivity” may represent a distinct pathophysiological abnormality in allergic eye disease [10]. The variability of symptoms experienced in allergic conjunctivitis which do not correlate with environmental changes such as the levels of sensitizing allergens, as well as the ocular reaction induced by non-sensitizing stimuli, may well be explained by this non-specific hyperreactivity. Natural non-specific stimulation with agents such as wind, dust, and sunlight may act only as